Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

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Calcineurin, or PP2B, the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain undiscovered. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal/ciliary proteins. These include POC5, which binds centrin in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT-type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin co-localizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes primary cilia elongation without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in cilia length maintenance, a process frequently disrupted in ciliopathies.

Calcineurin, or PP2B, the Ca2+ and calmodulin- activated phosphatase and target of immunosuppressants, has many substrates and functions that remain undiscovered. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal and ciliary proteins. These include POC5, which binds centrin in a Ca2+ -dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT-type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and expansion microscopy, we demonstrate that calcineurin co-localizes with POC5 at the centrosome, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centrosomal proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes primary cilia elongation without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in cilia length maintenance, a process frequently disrupted in ciliopathies.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. To discover novel calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a new calcineurin-regulated signaling pathway highlighting the PI4KA complex as a regulatory target and revealing that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

Calcineurin (CN), the serine/threonine protein phosphatase and the target of immunosuppressants FK506 and CysA, is a key regulator of Ca2+ signaling with critical roles in the immune, cardiovascular and nervous systems. CN is a heterodimer of regulatory and catalytic subunits whose functions and regulation by Ca2+ and calmodulin are well understood for canonical CN isozymes (CNalpha, CNβ2). In contrast, the CNβ1 isozyme, contains a catalytic subunit with a non‐canonical C‐terminus generated by alternative 3’ pre‐mRNA processing. The few studies of CNβ1, conserved in eukaryotes and broadly expressed in human tissues, demonstrate its unique physiological functions. For example, overexpression of the CNβ2 promotes cardiac hypertrophy through activation of NFAT, whereas CNβ1 is cardioprotective and does not dephosphorylate NFAT. However, CNβ1 specific substrates remain unknown. We determined that the unique C‐tail confers distinct regulatory properties, intracellular localization and function to CNβ1. In vitro, CNβ1 displays distinct enzymatic properties. Instead of the auto‐inhibitory domain that blocks the active site of canonical CN isoforms under non‐signaling conditions, CNβ1 is autoinhibited by a sequence motif at its C` tail that blocks substrate binding. We show that CNβ1 localizes to the plasma membrane (PM), Golgi, and intracellular vesicles, in contrast to the cytosolic CNβ2, due to the palmitoylation of two conserved cysteine residues unique to its C‐tail. Palmitoylation allows CNβ1 to access substrates that are distinct from canonical CN isoforms. CNβ1 preferentially interacts with membrane proteins, and all members of a highly conserved PI4‐kinase complex, PI4KIIIA, TTC7B, FAM126A and EFR3B, were identified as CNβ1‐specific interactors by AP‐MS. This complex recruits the cytosolic PI4KIIIA to the PM where it synthesizes phosphatidylinositol‐4‐phosphate (PI4P), a precursor of the critical signaling phospholipid, PI(4,5)P2, required for sustained Ca2+ signaling through GPCRs. We identify a CN binding motif in FAM126A, mutation of which results in FAM126A hyperphosphorylation, and prevents association of CNβ1 with the PI4KIIIA complex. Using BRET‐based detection of phosphoinositides in live cells, we show that CN promotes PI4P synthesis at the PM during signaling through the muscarinic receptor, and hypothesize that CNβ1 mediates this regulation by dephosphorylating FAM126A at the PM. Using pulse‐chase analysis in cells metabolically labelled with a palmitate analog, we demonstrate that palmitoylation of CNβ1 is dynamic and turns over rapidly. We are currently investigating whether dynamic palmitoylation regulates localization and activity CNβ1 activity in vivo during signaling. Together, these studies provide the first mechanistic understanding of CNβ1 and not only uncovers a novel Ca2+‐dependent mechanism for activation of PI4P synthesis at the PM during signaling but also suggests palmitoylation as a novel mechanism that confers spatio/temporal regulation of calcineurin signaling in cells. Support or Fu...